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Compositions and methods for transepithelial molecular transport

a technology of molecular transport and composition, applied in the direction of drug composition, immunological disorders, peptides, etc., can solve the problems of flaccid paralysis of the muscle, no vaccine that prevents botulism, and no research on botulism

Inactive Publication Date: 2009-02-26
THOMAS JEFFERSON UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the cytosol, BoNT blocks neuronal release of acetylcholine at the neuromuscular junction, causing flaccid paralysis of the muscle.
However, those molecules require production or isolation of intact HC, which has proven impractical for various reasons.
However, none describes vaccines that prevent botulism which are capable of transcytosing across epithelia.

Method used

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  • Compositions and methods for transepithelial molecular transport
  • Compositions and methods for transepithelial molecular transport
  • Compositions and methods for transepithelial molecular transport

Examples

Experimental program
Comparison scheme
Effect test

example 1

Detection of Transcytosis of BoNT A from the Apical Surface of T-84 Cells to the Basolateral Side of the Cells by Western Blotting

[0354]This experiment was carried out using native BoNT A and human gut epithelial cells (“T-84 cells”). Transcytosis was assayed in T-84 cell cultures using a TRANSWELL® apparatus assay system. Assay was initiated by addition of 1×10−8 molar native, purified, BoNT A to the upper chamber. Cultures were subsequently incubated for eighteen hours at 37° C. At the end of each experiment, contents of three basal chambers per condition were collected and concentrated in a CENTRICON™ micro-concentrator. The resulting solution was run on 7.5% (w / v) SDS-PAGE and subsequently transferred to nitrocellulose membranes. The identity and molecular weight of the transcytosed molecule was confirmed by Western blotting with anti-HC antibody.

[0355]A Western blot demonstrated that pre-transcytosis control (BoNT A) and BoNT A transcytosed through T-84 cells (i.e., collected f...

example 2

Western Blot of Apical to Basolateral Transcytosed BoNT A in MDCK Cells

[0356]This experiment was carried out using BoNT A and Madin-Darby Canine Kidney Cells (“MDCK cells”). Transcytosis was assayed in MDCK cell cultures using a TRANSWELL® apparatus assay system. Assay was initiated by addition of 1×10−8 molar native, purified, BoNT A to the upper chamber. Cultures were subsequently incubated for eighteen hours at 37° C. At the end of each experiment, contents of three basal chambers per condition were collected and concentrated in a CENTRICON™ micro-concentrator. The resulting solution was run on 7.5% (w / v) SDS-PAGE and subsequently transferred to nitrocellulose membranes. The identity and molecular weight of the transcytosed molecule was confirmed by Western blotting with anti-HC antibody.

[0357]The Western blot indicated that native BoNT A is poorly bound, internalized, transcytosed, and released by MDCK cells. BoNT A was not detected in medium collected from the basolateral side ...

example 3

Apical to Basolateral Transcytosis of BoNT A in T-84 Cells

[0358]Transcytosis of BoNT A linked to 125I at lysine residues was assayed in human gut epithelial cells (“T-84”) cell cultures using a TRANSWELL® apparatus assay system. Assay was initiated by addition of 1×10−8 molar (125I)-BoNT A to the upper chamber. Cultures were subsequently incubated for eighteen hours at 37° C. At the end of each experiment contents of the basal chamber were collected and gel filtered. Void volume fractions were assayed for radioactivity and the toxin peak was summed to determine total counts. The amount of transcytosis was calculated based on the specific activity of labeled BoNT A.

[0359]The results indicate that BoNT A linked to 125I was transported from the apical to the basolateral side of cells. It efficiently crossed T-84 cells at a transcytosis rate of 11.29±0.30 femtomoles per hour per square centimeter.

[0360]There are three major conclusions that stem from the experimental results. First, the...

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Abstract

The invention relates to fragments of Clostridium botulinum HC that can be linked with an entity (e.g., an antigen, a particle, or a radionuclide) and used to deliver the entity across a non-keratinized epithelial membrane of an animal. The fragments are useful, for example, for making vaccines, antidotes, and anti-toxins and in situations in which rapid uptake of an agent by an animal is desired.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional application of copending U.S. patent application Ser. No. 10 / 452,024, filed on Jun. 2, 2003, which application claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application Ser. No. 60 / 384,949, filed May 31, 2002, the contents of each which are incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This research was supported in part by U.S. Government funds (National Institutes of Health grant number GM057342).INCORPORATION BY REFERENCE OF MATERIAL ON COMPACT DISK[0003]This application incorporates by reference the Sequence Listing contained on the two compact disks (Copy 1 and Copy 2), filed on Jan. 11, 2007, in the copending parent U.S. patent application Ser. No. 10 / 452,024, and containing the following file: File name: 08321-0191US1 Substitute sequence listing.txt; created Jan. 3, 2007; 1,064,960 bytes in size.BACKGROUND OF THE INVENTION...

Claims

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Application Information

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IPC IPC(8): A61K39/385A61K39/395C07K16/00A61P37/04A23L1/30A61K38/00A61K39/00A61K39/02A61K39/07A61K39/08A61K39/10A61K39/106A61K39/12A61K39/193A61K39/215A61K47/42A61K47/48A61K49/04A61K51/00A61P31/04A61P31/12C07K14/33C12N5/07C12N5/078C12N5/079C12N5/10C12N15/09
CPCA61K39/385A61K47/48261A61K47/4833C12N2760/12222A61K2039/543A61K2039/544A61K2039/6037A61K2039/542A61K47/6415A61K47/646A61P31/04A61P31/12A61P37/04Y02A50/30
Inventor SIMPSON, LANCEMAKSYMOWYCH, ANDREWPARK, JONG-BEAK
Owner THOMAS JEFFERSON UNIV
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